The productivity of plants is limited by the three primary nutrients: nitrogen, phosphorous and potassium, in most natural and agricultural ecosystems. Generally nitrogen is the most important of the three limiting nutrients and the major component in fertilizers. Since nitrogen is usually the rate-limiting element in plant growth, most field crops have a fundamental dependence on inorganic nitrogenous fertilizer. The nitrogen source in fertilizer is usually ammonium nitrate, potassium nitrate, or urea.
Increased nitrogen use efficiency by plants has a number of beneficial effects, for example, increased growth and yield when compared to conventional plants grown in nitrogen poor soils, and reduced requirement for the addition of nitrogenous fertilizers to crops. Fertilizers account for a significant percentage of the costs associated with crop production, therefore using less fertilizer would reduce the producers' costs. A reduction in fertilizer application would also lessen the environmental damage resulting from extensive nitrogenous fertilizer use. Excess fertilizer application causes increased eutrophication, acid rain, soil acidification and the greenhouse effect. These environmental disasters cause further problems such as fish kills, loss of biodiversity, increased algal blooms, loss of arable land and accelerated global climate change, affecting the world population on both social and economic scales.
Monocots represent a large percentage of the crops grown in the world with approximately 217 million hectares of wheat and 158 million hectares of both maize and rice planted in 2007. Approximately half of the global calorie and protein requirement is derived from wheat, rice and maize. Rice is routinely used as a model crop for genetic and physiological studies in other monocot crops including maize, wheat, sugarcane, barley, sorghum, rye and grass. Rice has a small, diploid genome that is well conserved and syntenic across monocots.
Promoters are nucleic acid sequences that allow for regulation of transcription of a gene or nucleotide sequence. Promoters can allow for constitutive expression, such as the well known cauliflower mosaic virus promoter CaMV 35S, inducible expression, such as the stress inducible promoter rd29A (Pino et al., 2007), tissue specific expression, such as root specific OsANT1 (U.S. Patent Application Publication No. 2009/0288224) and developmentally specific expression, such as the senescence induced IPT promoters (Ma et al., 2009). Promoters can also be weak or strong, suggesting that whenever or wherever they are induced, they will allow for expression of the attached gene or nucleotide sequence at varying levels.
When a promoter is fused to the 5′ end of a gene or nucleotide sequence, it can regulate the expression of that gene or nucleotide sequence. However not all promoters will successfully express all genes or nucleotide sequences in all plant types. For example, a dicot promoter may not perform in the same manner when placed in a monocot system, and vice versa. Similarly, a monocot promoter may not function in the same manner when placed in a monocot of a different Genus, such as a rice promoter placed into wheat. For transgenic studies, there are different types of promoters that can be used, depending on the goal of the experiment. Promoters are often classified as constitutive, tissue specific and/or inducible. Many transgenic studies to date have used generic constitutive promoters such as the cauliflower mosaic virus (CAMV35S) and maize ubiquitin 1 promoter (ubi-1) to drive target gene over-expression in plants. This can be a disadvantage as it could be energetically unfavorable for plants to express the gene at all times and it could produce abnormal development, since expression levels of the transgene is not regulated (Shelton et al., 2002). For example, constitutive over-expression of the cellulose synthase like gene CslF6 by the oat globulin promoter ProASGL frequently resulted in reduced germination of seeds or seedlings with necrosis on the leaf tips leading to death in severe cases (Burton et al., 2011).
Using inducible or tissue specific promoters may be a better option to drive transgene expression. Inducible promoters will only drive gene expression when a specific physical, environmental, biological or chemical stimulus is applied. The heat inducible promoter of the Hvhsp17 gene from wheat can be used for high target gene expression when plants are exposed to 38 to 40° C. for 1 to 2 hours (Freeman et al., 2011). This allows for the short term gene or nucleotide sequence expression and control of developmental expression but is limited to tissues that are not severely affected by temperature changes. Over-expression of the Triticum aestivum NAC protein (TaNAC69), encoding a transcription factor involved in drought stress by the drought inducible promoter HvDhn4s, produced wheat plants with significantly higher shoot biomass at the early vegetative stage under mild salt stress and water limitation, compared to the wild-type. Conversely, the HvDhn8s constitutive promoter driving TaNAC69 showed no significant difference from untransformed controls (Xue et. al., 2011). When the drought and cold stress inducible promoter rdA29 was used to over-express the DREB1A gene, which encodes a transcription factor involved in stress tolerance in Arabidopsis, normal plants were generated, while constitutive expression driven by the CAMV35S promoter resulted in growth retardation under normal growing conditions (Kasuga et al., 1999).
Tissue specific promoters are involved in organ specific and developmental stage specific expression of transgenes. For example, in potatoes, StRCAp is engineered into the leaves as a defense mechanism against predatory insects, but is not expressed in the eating parts of the plant that produces this toxin (Weber, 2003; Park and Jones, 2008). The use of the 35S promoter has also raised concerns of food safety where the toxin produced in non-target organs might cause the potatoes to be unsafe for consumption. In addition, it may be metabolically taxing for the plant to be constantly producing a secondary metabolite, regardless of the developmental stage or organ, therefore causing plants to be less healthy and potentially compromising yields.
In the case of NUE plant engineering, tissue specific expression of genes might increase the efficacy of N uptake, utilization or remobilization in the plant. In contrast, the use of a constitutive promoter might prove to be a waste of energy because over-expression of non-rate limiting enzymes in certain organs may not produce any phenotype or may even decrease yield. Development of an NUE plant may also involve the transformation of multiple genes or gene stacking to achieve a satisfactory NUE phenotype, since N metabolism and transport are very complex processes.
In addition, the use of the CAMV35S promoter may not produce any phenotype because the gene expression or protein expression was not sufficient in a specific organ or developmental phase. Also, plants could turn off the expression of the transgene when it proves to be energetically unfavourable. When HvAlaAT was driven by the CAMV35S promoter, it did not exhibit any NUE phenotype. However, when the root specific btg26 promoter was used, it produced plants that had higher NUE (Good et al., 2007).
Similar to over-expression of target genes, promoters when used in a different species may not mimic the expression patterns of its native species. Seed specific promoters from barley (B-hor and D-hor) and wheat (HMW-Glu) did not direct seed specific expression in rice, instead the promoters drove high expression levels in leaf, shoot and maternal seed tissues of rice plants (Wu et al., 1998; Qu and Takaiwa, 2004; Furtado et al., 2008).
PBpr1 promoters are from genes encoding methyl-melonate semialdehyde dehydrogenase (MMSDH). The PBpr1 promoter is upstream of the OsALDH6 gene, which encodes a gene for methylmalonate semialdehyde dehydrogenase in rice (Accession number: gene: AK 121280.1 and mRNA: AF045770.1). AK121280.1 and AF045770.1 are splice variants of each other where AF045770 is shorter than the AK121280.1. AK121280.1 appears to be a hypothetical protein designated by GenBank, while AF045770.1 has been characterized (Oguchi et al (2004). OsALDH6 is homologous to the ALDH6B2 gene in Arabidopsis, which also encodes for a methlymalonate semialdehyde dehydrogenase. The OsALDH6 gene is highly expressed in young roots and stems (Gao and Han, 2009).
Methylmalonate semialdehyde dehydrogenase (MMSDH) catalyzes the irreversible oxidative decarboxylation of malonate semialdehydes to acetyl-CoA and methylmalonate-semialdehyde to propionyl-CoA in the distal portions of the valine and pyrimidine catabolic pathways. Since MMSDH generates acetyl-CoA, it is an important factor in the glyoxylate pathway, TCA cycle and fatty acid production.
MMSDH is down-regulated during oxidative stress due to the restriction in the TCA cycle and the production of ATP (Sweetlove et al., 2002). In two week old rice plants, MMSDH mRNA was found at high levels in roots and leaf sheaths while protein accumulation was found highest in roots followed by leaf blades (Oguchi et al., 2004). With the addition of auxin, MMSDH levels in roots increased along with an increase in rooting. Tanaka et al. (2005) has suggested that MMSDH is involved in root growth, tissue differentiation and thickening growth due to its expression in crown roots, lateral roots and root hairs. MMSDH was found to be localized in the mitochondrial matrix of Arabidopsis, rice, human, bovine and rats which suggests a similarity of function of MMSDH among all these organisms.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.